Sites of p-chloromercuribenzenesulfonate inhibition of red cell urea and water transport

The mercurial sulfhydryl reagent, p-chloromercribenzene sulfonate (pCMBS) inhibits water and urea fluxes across the human red blood cell membrane. The kinetics and affinities for pCMBS binding to separate water transport and urea transport inhibition sites were previously determined by Toon and Solomon ((1986) Biochim. Biophys. Acta 860, 361-375) in red cells that had been treated with N-ethylmaleimide (NEM) to block five of the six sulfhydryls on the red cell anion exchange protein, band 3. We have used autoradiographs of gels from NEM-treated cells, labeled with ²⁰³Hg-pCMBS, to localize these water and urea transport inhibition binding sites separately and find that both are on band 3. Each site is saturable and the time course of each uptake can be fitted to the equation for a bimolecular associations (with negligible dissociation) with time constants in agreement with those of Toon and Solomon. Determination of the binding stoichiometry shows one urea inhibition site and three water inhibition sites for every four band 3 molecules. These results indicate that band 3 plays a role in both urea and water transport and suggest that we functional unit may be a tetramer.